Photosynthetic Efficiency of Marchantia polymorpha L. in Response to Copper, Iron, and Zinc

Metal micronutrients are essential for plant nutrition, but their toxicity threshold is low. In-depth studies on the response of light-dependent reactions of photosynthesis to metal micronutrients are needed, and the analysis of chlorophyll a fluorescence transients is a suitable technique. The liverwort Marchantia polymorpha L., a model organism also used in biomonitoring, allowed us to accurately study the effects of metal micronutrients in vivo, particularly the early responses. Gametophytes were treated with copper (Cu), iron (Fe) or zinc (Zn) for up to 120 h. Copper showed the strongest effects, negatively affecting almost the entire light phase of photosynthesis. Iron was detrimental to the flux of energy around photosystem II (PSII), while the acceptor side of PSI was unaltered. The impact of Fe was milder than that of Cu and in both cases the structures of the photosynthetic apparatus that resisted the treatments were still able to operate efficiently. The susceptibility of M. polymorpha to Zn was low: although the metal affected a large part of the electron transport chain, its effects were modest and short-lived. Our results may provide a contribution towards achieving a more comprehensive understanding of response mechanisms to metals and their evolution in plants, and may be useful for supporting the development of biomonitoring techniques.


Introduction
Heavy metal pollution is a growing concern for both human and ecosystems' health. Natural processes release these elements from the Earth's crust, but anthropogenic activities are responsible for dispersing large amounts of them worldwide [1,2]. The distinctive feature of heavy metals is their density, which exceeds 5 g cm −3 [3]. Beyond being very persistent in the environment, most of them are toxic for all living organisms [4,5]. Some heavy metals are instead essential for plant nutrition if acquired in small levels, otherwise they can easily become harmful when absorbed in amounts exceeding the plant's physiological needs [6]: in this case, they may also induce the overproduction of reactive oxygen species (ROS), which target key biological molecules [2]. Further negative effects of heavy metals are osmotic stress, plasmolysis, extreme vacuolation of cells and accumulation of excess starch [4,[7][8][9].
Several heavy metals also adversely affect photosynthesis, in diverse ways. These elements may lower the number and size of chloroplasts and disrupt thylakoid arrangement [10][11][12][13]. Stomatal and mesophyll conductance for CO 2 decline in the leaves of plants growing in heavy metal-contaminated soils [14,15]. The concentration of photosynthetic pigments decreases following exposure to heavy metals, owing to chlorophyll biosynthesis repression; moreover, chlorophyll function may be impaired by the substitution of Mg in the center of the porphyrin ring [16][17][18]. The whole photosynthetic electron transport chain can be severely affected by heavy metals [15] and photosystem II (PSII) is particularly vulnerable. Both the donor and the acceptor side of PSII may be damaged by excessive concentrations of some metal nutrients like, for example, Cu and Zn [19][20][21][22].
(Marchantiophyta) represents a crucial step in plant phylogeny [28,29] and is a valuable model organism in functional, molecular, and evolutionary studies on land plants, as well as an excellent tool for biomonitoring [30,31]. Nevertheless, there is limited knowledge about the responses of M. polymorpha to regulate and cope with high concentrations of metal micronutrients. In recent years, an article has shed light on the effects of elevated toxicity in Marchantia polymorpha when exposed to high concentrations of Cu and Zn (0.2 and 2 mM) over a long exposure time [31]. To help bridge this knowledge gap, our work is aimed at investigating in depth the response of M. polymorpha photosynthesis light reactions to excessive amounts of some metal micronutrients, namely Cu, Zn and Fe. Experimental evidence on this topic is scant, but it is crucial to achieve a thorough understanding of the mechanisms involved in metal detoxification, for the development of effective biomonitoring techniques that rely on the application of this species in environmental studies with heavy metals. Moreover, knowledge of physiological responses of photosynthesis to metal micronutrients may pave the way to biochemical and molecular studies on the evolution of metal tolerance in land plants. The outcome of exposure to heavy metals may be effectively investigated by analyzing ChlF, which is particularly suitable for revealing stress responses right from the early stages.

Results
The effects of treatments with excess heavy metals have been quantified using the parameters described in Table 1, most of which are the results of JIP test. The effects of each metal at each concentration and for each treatment time were compared with the respective controls. Below are described only the results related to the metal-concentration-time combinations that had significant effects. Data interpretation is based mostly on [32][33][34][35]. The subscript letter following "CS" means that the parameter refers to the state of all RCs open (subscript "O") or to the state of all RCs closed (subscript "M"). The treatment 200 µM Cu (Cu200) resulted in a decrease in energy absorption by the PSII antenna (ABS/CS M ) and in energy transfer from Q A − to the intersystem electron acceptors (ET 0 /CS M ) when all RCs were closed. Furthermore, there was a lower energy trapping by active PSII units (TR 0 /CS O and TR 0 /CS M ), i.e., the reduction of Q A was lessened (both when all RCs were open and when they were all closed).

Responses to Excess Cu
The treatment Cu200 induced changes in the pattern of fluorescence transients after 14 h (Figure 1c) and broad negative effects were revealed by JIP test (Figure 1d), which demonstrated the alteration of many parameters belonging to all the main groups illustrated in Table 1.
The lower values of ABS/CS O (≈F O ) suggest a diminished absorption of photon energy per excited PSII cross section with open RCs, leading to weaker fluxes of energy dissipation (DI 0 /CS O , but it was evident also when RCs were closed: see the value of DI 0 /CS M ) and trapping (TR 0 /CS O ). There was a decrease in the number of active RCs (RC/CS O ); consequently, their turnover number (N, i.e., the rate of their reduction and re-oxidation) declined. The contraction of the number of active RCs is substantiated by the lower value of Area, which is a proxy of the number of Q A acceptors, and the higher value of V J , that provides an estimation of the number of closed RCs, i.e., of reduced Q A molecules. The energy flux from Q A − to the intersystem electron acceptors was severely affected in treated gametophytes, as demonstrated by many parameters (ET 0 /RC, ET 0 /CS O and ET 0 /CS M ). Furthermore, the efficiency with which a PSII trapped electron was transferred from Q A − to the secondary quinone acceptor (Q B or, particularly when it is not bound to PSII, plastoquinone, PQ) (ψE 0 ), as well as the quantum yield of such transfer (ϕE 0 ), were lessened by the treatment. The value of the performance index ψ(E 0 )/(1 − ψ(E 0 )) shows that the contribution of intersystem electron transport to the global performance of photosynthesis light reactions was negatively affected by the exposure to 200 µM Cu. Adverse effects were detected also on the transfer of energy from Q A − to the end acceptors of PSI, both per active PSII (RE 0 /RC) and per excited PSII cross section (RE 0 /CS O and RE 0 /CS M , respectively). The lower values of ABS/CSO (≈FO) suggest a diminished absorption of photon energy per excited PSII cross section with open RCs, leading to weaker fluxes of energy dissipation (DI0/CSO, but it was evident also when RCs were closed: see the value of DI0/CSM) and trapping (TR0/CSO). There was a decrease in the number of active RCs (RC/CSO); consequently, their turnover number (N, i.e., the rate of their reduction and re-oxidation) declined. The contraction of the number of active RCs is substantiated by the lower value of Area, which is a proxy of the number of QA acceptors, and the higher value of VJ, that provides an estimation of the number of closed RCs, i.e., of reduced QA molecules. The energy flux from QA − to the intersystem electron acceptors was severely affected in treated gametophytes, as demonstrated by many parameters (ET0/RC, ET0/CSO and ET0/CSM). Furthermore, the efficiency with which a PSII trapped electron was transferred from QA − to the secondary quinone acceptor (QB or, particularly when it is not bound to PSII, plas-  Table 1), normalized to the values of the control, which were set as one. Black lines = control; red lines = 200 µM Cu; orange line = 80 µM Cu. Only those parameters that differed significantly from the control (p < 0.05) are shown. All values are the mean of nine replications.
Exposure of gametophytes for 24 h to 80 µM Cu (Cu80) yielded significant effects, as suggested by the pattern of ChlF ( Figure 1e) and JIP test (Figure 1f). The treatment Cu80 diminished the number of RCs, as shown by the decrease of Area, that led to a fast accumulation of reduced Q A (higher V J and (∆V/∆t) 0 ). Treated gametophytes partially lost their efficiency of transferring the trapped energy from Q A − to Q B (decreased ψE 0 and ϕE 0 ), which conceivably lowered the performance of intersystem electron transport (ψ(E 0 )/(1 − ψ(E 0 ))). The decrease of the performance index of energy conservation of After 24 h, Cu200 also altered substantially the light reactions of photosynthesis. Fluorescence transients were not notably upset (Figure 1g), while JIP test (Figure 1h) highlighted several consequences of the treatment. Damage to the oxygen-evolving complex of PSII (OEC) was evident from the high value of V K . Impairments in RCs were demonstrated by the low Area, and high V J plus (∆V/∆t) 0, which means fewer total RCs and a greater number of reduced RCs. Energy flux in the intersystem declined (lower ET 0 /CS M ) and became less efficient (decreased ψE 0 and ϕE 0 ). These alterations had a negative impact on the two performance indexes of energy conservation of absorbed photons (PI ABS and PI tot ), pointing at a general decline of the photosynthetic process. The rise of ABS/RC seems to reveal an increase in the apparent antenna size of active PSII, but actually the real cause is the decrease in the number of active The contributions of primary photochemistry reactions (ϕ(P 0 )/(1 − ϕ(P 0 ))) and of intersystem electron transport (ψ(E 0 )/(1 − ψ(E 0 ))) to the global performance of photosynthesis light reactions were diminished by the Cu treatment, with negative consequences on the performance indexes of energy conservation of absorbed photons (PI ABS and PI tot ).

Responses to Excess Fe
Exposure of M. polymorpha for 6 h to Fe induced a significant response only at 300 µM concentration (Fe300). Compared to the control, the ChlF emission differed only slightly and JIP test revealed only one significant difference between Fe300 and the control (Figure 2a Table 1); values in spider plots were normalized to those of the control, which were set as one. Black lines (or bars) = control; red lines (or bars) = 300 µ M Fe. Only those parameters that differed significantly from the control (*, p < 0.05) are shown. All values are the mean of nine replications.
The treatment 200 µ M Fe (Fe200) started to cause significant effects at 24 h. Fluorescence transients (Figure 3a) diverged from that of the control and JIP test (Figure 3b) showed that the efficiency (ψE0) and the quantum yield (φE0) of electron transfer from QA − to QB were lower in Fe200 than in control, as well as the contribution of intersystem electron transport to the global performance of photosynthesis light reactions (ψ(E0)/(1−ψ(E0))). The slowdown of electron transport in the intersystem might be the cause of the accumulation of closed RCs, evidenced by high VJ, and of the reduction of the performance index up to QB reduction (PIABS).

Figure 2.
Effects of the exposure to 300 µM Fe for 6 (a,b), 14 (c,d), 24 (e,f), 72 (g,h) and 120 h (i,j), in dark-adapted M. polymorpha gametophytes. Induction transients of ChlF (a,c,e,g,i) and spider plots (d,h,j) or bar charts (b,f) of parameters of JIP test (described in Table 1); values in spider plots were normalized to those of the control, which were set as one. Black lines (or bars) = control; red lines (or bars) = 300 µM Fe. Only those parameters that differed significantly from the control (*, p < 0.05) are shown. All values are the mean of nine replications.
Also, at 14 h the sole effective treatment was Fe300. Raw ChlF data of control and treated samples were similar (Figure 2c , the efficiency of primary photochemistry of PSII was enhanced (higher F V /F M and ϕP 0 ). The active RCs may have benefited from the diminished dissipation of energy. It is reasonable to believe that, for the same reason, primary photochemistry has made a greater contribution to overall performance (higher ϕ(P 0 )/(1 − ϕ(P 0 ))).
At 24 h, the gametophytes Fe300 showed a ChlF emission almost coincident with that of the control ( After 120 h, the effects on Fe300 gametophytes were more evident: the curve of ChlF emission appeared to diverge more widely from that of the control (Figure 2i) and JIP test revealed several differences between Fe300 and control samples (  (Figure 3b) showed that the efficiency (ψE 0 ) and the quantum yield (ϕE 0 ) of electron transfer from Q A − to Q B were lower in Fe200 than in control, as well as the contribution of intersystem electron transport to the global performance of photosynthesis light reactions (ψ(E 0 )/(1 − ψ(E 0 ))). The slowdown of electron transport in the intersystem might be the cause of the accumulation of closed RCs, evidenced by high V J , and of the reduction of the performance index up to Q B reduction (PI ABS ).  Table 1); values in spider plot were normalized to those of the control, which were set as one. Black line (or bar) = control; orange line (or bar) = 200 µ M Fe. Only those parameters that differed significantly from the control (*, p < 0.05) are shown. All values are the mean of nine replications.
The treatment Fe200 at 72 h exhibited a ChlF emission similar to the control ( Figure  3c) and JIP test highlighted only one significant difference (Figure 3d), namely a lower t for FM in treated gametophytes: this indicated that the maximal fluorescence FM was reached faster, due to a reduced capacity to transport electrons.

Responses to Excess Zn
The overall effect of the exposure to Zn was rather mild. Gametophytes treated with 80 µ M of the metal (Zn80) exhibited significant effects only at 72 h. Fluorescence transients of Zn80 samples were very similar to the control (Figure 4a), and JIP test (Figure 4b) revealed limited differences. Treated gametophytes accumulated reduced QB (higher VI) and the electron transport on the acceptor side of PSI was negatively affected, both per excited cross section of PSII (lower RE0/CSO) and as quantum yield (φR0). The latter seems to be attributable to the lower efficiency with which an exciton trapped by PSII is transferred to final PSI acceptors (ψR0).  Table 1); values in spider plot were normalized to those of the control, which were set as one. Black line (or bar) = control; orange line (or bar) = 200 µM Fe. Only those parameters that differed significantly from the control (*, p < 0.05) are shown. All values are the mean of nine replications.
The treatment Fe200 at 72 h exhibited a ChlF emission similar to the control (Figure 3c) and JIP test highlighted only one significant difference (Figure 3d), namely a lower t for F M in treated gametophytes: this indicated that the maximal fluorescence F M was reached faster, due to a reduced capacity to transport electrons.

Responses to Excess Zn
The overall effect of the exposure to Zn was rather mild. Gametophytes treated with 80 µM of the metal (Zn80) exhibited significant effects only at 72 h. Fluorescence transients of Zn80 samples were very similar to the control (Figure 4a), and JIP test (Figure 4b) revealed limited differences. Treated gametophytes accumulated reduced Q B (higher V I ) and the electron transport on the acceptor side of PSI was negatively affected, both per excited cross section of PSII (lower RE 0 /CS O ) and as quantum yield (ϕR 0 ). The latter seems to be attributable to the lower efficiency with which an exciton trapped by PSII is transferred to final PSI acceptors (ψR 0 ). 80 µ M of the metal (Zn80) exhibited significant effects only at 72 h. Fluorescence transients of Zn80 samples were very similar to the control (Figure 4a), and JIP test (Figure 4b) revealed limited differences. Treated gametophytes accumulated reduced QB (higher VI) and the electron transport on the acceptor side of PSI was negatively affected, both per excited cross section of PSII (lower RE0/CSO) and as quantum yield (φR0). The latter seems to be attributable to the lower efficiency with which an exciton trapped by PSII is transferred to final PSI acceptors (ψR0).   Table 1); values in spider plot were normalized to those of the control, which were set as one. Black line (or white bar) = control; pale blue line = 80 µM Zn; dark blue bar = 200 µM Zn. Only those parameters that differed significantly from the control (*, p < 0.05) are shown. All values are the mean of nine replications.
Significant, yet limited, effects were observed in gametophytes treated with 200 µM Zn (Zn200) after 24 h. The pattern of ChlF emission (Figure 4c) differed between treated samples and control, but JIP test (Figure 4d) demonstrated that only PI ABS was significantly different: the value was lower in treated samples, thus indicating a lower performance of energy conservation of absorbed photons up to Q B reduction.

Overall Impact of the Treatments
The treatments that produced the strongest effects on the light reactions of photosynthesis were Cu200 and Fe300. The effects of Zn were small, at both concentrations applied. The Cu80 and Fe200 treatments had minor effects, that were evident particularly after 24 h: both metals decreased the efficiency of electron transport in the intersystem and, in the case of Cu, also the electron flux in the acceptor side of PSI. The effects of Cu200 and Fe300 were summarized in Figures 5 and 6, respectively.
The effects of Cu200 ( Figure 5) were already evident after 6 h and appeared quite severe at 14 h. At 24 h, there seemed to be a slight recovery (as far as concerned electron flux per excited cross section of PSII), but the situation worsened again afterward (technical parameters, phenomenological energy fluxes and performance indexes). The efficiencies/quantum yields and the specific energy fluxes per active RC did not show critical values. However, after 120 h, problems began to arise also in OEC, and energy dissipation increased. At this concentration, Cu strongly reduced the flow of electrons throughout the transport chain, as well as photon absorption in the antennas.
In general, Fe300 ( Figure 6) caused less severe effects than Cu200. A recovery at 24 h was apparent in Fe300 (only one parameter of JIP test was worse than the control), followed by a worsening of conditions, as indicated by the values of phenomenological energy fluxes. Modest effects were recorded on quantum efficiencies/yields and specific energy fluxes per RC. At 14 h, there were even some positive impacts on OEC, although they were transient. Part of the RCs were inactivated (as demonstrated by the decrease in RC/CS values) and the photon absorption in the antennas decreased, as well as the flow of electrons in the photosynthetic transport chain; however, the acceptor side of PSI was almost unaffected.

Overall Impact of the Treatments
The treatments that produced the strongest effects on the light reactions of photosynthesis were Cu200 and Fe300. The effects of Zn were small, at both concentrations applied. The Cu80 and Fe200 treatments had minor effects, that were evident particularly after 24 h: both metals decreased the efficiency of electron transport in the intersystem and, in the case of Cu, also the electron flux in the acceptor side of PSI. The effects of Cu200 and Fe300 were summarized in Figures 5 and 6, respectively. The effects of Cu200 ( Figure 5) were already evident after 6 h and appeared quite severe at 14 h. At 24 h, there seemed to be a slight recovery (as far as concerned electron flux per excited cross section of PSII), but the situation worsened again afterward (technical parameters, phenomenological energy fluxes and performance indexes). The efficiencies/quantum yields and the specific energy fluxes per active RC did not show critical values. However, after 120 h, problems began to arise also in OEC, and energy dissipation increased. At this concentration, Cu strongly reduced the flow of electrons throughout the transport chain, as well as photon absorption in the antennas. In general, Fe300 ( Figure 6) caused less severe effects than Cu200. A recovery at 24 h was apparent in Fe300 (only one parameter of JIP test was worse than the control), followed by a worsening of conditions, as indicated by the values of phenomenological energy fluxes. Modest effects were recorded on quantum efficiencies/yields and specific energy fluxes per RC. At 14 h, there were even some positive impacts on OEC, although they were transient. Part of the RCs were inactivated (as demonstrated by the decrease in RC/CS values) and the photon absorption in the antennas decreased, as well as the flow Figure 6. Heat map representing relative variability of the analyzed photosynthesis-related parameters, following treatment of M. polymorpha gametophytes with 300 µM Fe. Red is for lower values and green for the highest values. All data were first normalized to bring the value of the parameters in the range 1-100.

Discussion
Photosynthetic light reactions are particularly vulnerable to the action of heavy metals, which can cause wide-ranging effects such as thylakoid membrane disorganization, damage to OEC and impaired electron transport [8,36,37]. Using ChlF analysis, a key non-invasive technique for studying the photosynthetic apparatus in vivo, our work has shown that also in the liverwort M. polymorpha excessive concentrations of metal micronutrients can have significant negative impacts on photosynthesis.
The strongest effects were shown by Cu, thus substantiating the inhibition of photosynthesis by this metal that had been previously observed in some higher plants and algae [38,39]. According to the literature, Cu appears to inhibit photosynthetic electron transport, with the acceptor and donor sides of PSII indicated as the most sensitive targets [40][41][42][43]. Our study confirmed such results and added some further details. The treatment Cu200 negatively affected almost all processes of the light phase of photosynthesis, starting from the absorption of photons, up to the electron flow on the acceptor side of PSI. This metal is known to inhibit pigment accumulation, to replace Mg within the chlorophyll molecule and to hinder its integration into the photosystems. Furthermore, Cu can induce the release of proteins from inner antenna (CP47 and CP43) of PSII [44] and can inhibit the reduction of Q A and Q B [45]. The negative effects of Cu that we detected on electron transport in the acceptor side of PSI might be explained by the interference of the metal with ferredoxin, as previously observed in spinach [46]. In addition, when present in high concentrations and for prolonged periods of time, Cu can cause the closure also of PSI [47], beyond that of PSII. In M. polymorpha, the efficiencies/quantum yields and the specific energy fluxes per active RC did not attain critical values, suggesting that the components of the photosynthetic apparatus that had remained active were still functioning fairly efficiently, although they were exposed to high Cu concentrations. Despite this, at the end of the experiment the phenomenological energy fluxes displayed a marked decline of photosynthetic efficiency in M. polymorpha. Furthermore, OEC was negatively affected, owing probably to the known ability of Cu to inhibit the Mn cluster and/or the tyrosine TyrZ or TyrD residues [40].
The effects of Cu were far more evident at the higher concentration applied, i.e., 200 µM. The response to 80 µM Cu was much weaker and of shorter duration, and was detected only at 24 h. It altered mainly the number and activity of RCs, and the efficiency of the intersystem electron transport. In maize leaves, the same Cu concentration caused denaturation of PSII, resulting in a significant decline of electron transport [47]. M. polymorpha appeared to be more resistant than maize and, surprisingly, Cu had also a slight positive effect on the electron flux in the acceptor side of PSI. The reason for this is not clear and it can only be hypothesized that such positive effect might be related to the fact that Cu, being a component of plastocyanin, is actively involved in photosynthetic electron transport. Low concentrations of Cu (up to 20 µM, applied for 24 h) exhibited some beneficial effect on photosynthesis also in Lemna minor [48].
The role of Fe in the photosynthetic process is well documented: this metal micronutrient, owing to its function in redox reactions, is a constituent of several complexes involved in electron transport and preserves the structure and function of RCs and antenna complexes. However, excess Fe may cause toxic effects that are mediated by ROS overproduction [49]. In M. polymorpha, JIP test showed a stronger and earlier impact of the treatment at the highest concentration, i.e., 300 µM, while the response to 200 µM Fe was significant only after 24 h and, albeit extremely weakly, at 72 h of exposure. The treatment Fe200 at 24 h accelerated the accumulation of closed RCs and decreased the flux of electrons from Q A − to Q B , while the transport in the acceptor side of PSI did not seem to be affected. The Fe300 gametophytes began to show negative effects well before Fe200. Similar to the latter, they suffered inhibition of electron transfer from Q A − to Q B and did not show negative consequences on energy fluxes and transport efficiency on the acceptor side of PSI, even after 120 h of treatment. Only at 72 h there was a decrease in the flux of electrons to final PSI acceptors per excited cross section of PSII, but it was transient and could have been an indirect consequence of the lower absorbed photon flux. Similar results were obtained in Ipomoea batatas L.: after exposure to 9 mM Fe for 7 days, there was a reduction in net photosynthesis, but also a positive effect of the treatment on some sections of the electron transport chain, including that on the acceptor side of PSI [50]. Other transient positive effects of Fe300 were recorded on primary photochemistry and OEC at 14 h, while lower dissipation of absorbed energy was observed at 14, 72 and 120 h. Despite that its impact was partially beneficial, Fe300 treatment had negative consequences that seemed to worsen over time, especially on photon absorption and electron flow around PSII, up to Q B , whereas the acceptor side of PSI was unaffected, and, after prolonged exposure, its transport efficiency was even enhanced. Overall, our data seem to confirm that Fe does not inhibit photosynthesis severely. On the contrary, at the highest concentration applied it also produced some beneficial effects, perhaps because this element is a fundamental constituent of many complexes of the photosynthetic apparatus, such as cytochromes and Fe-S clusters, as well as being an essential cofactor in the biosynthesis of these complexes and chlorophylls [51]. The impact of the treatment Fe300 was less strong than that of Cu200, but some common features were observed in the responses of gametophytes. In both cases a transient recovery occurred at 24 h, but the scenario worsened with increasing exposure times. A further common outcome of the two treatments was the mild effect on quantum efficiencies/yields and specific energy fluxes per RC: this apparently implies that the structures that kept functioning despite the treatments were still able to operate efficiently.
Zinc may have multiple negative effects on photosynthesis. Excess of this heavy metal causes reduction in photosynthetic pigments synthesis and damages the photosynthetic machinery [32]. This element replaces Mg in chlorophyll molecules [52] and may induce the release of three extrinsic polypeptides of OEC [44]. Additionally, in Phaseolus vulgaris, Zn has been shown to inhibit PSI and PSII and to negatively affect the synthesis of ATP [53]. Despite its potential toxicity, Zn did not exhibit all these negative effects on the gametophytes of M. polymorpha. The treatment Zn80 seemed to impact, only transiently (at 72 h), the electron transport beyond Q B , by reducing the energy flux per active PSII cross section and the efficiency of such transport, while leaving energy absorption and trapping almost unchanged. The treatment Zn200 had a negative effect only at 24 h, when it reduced the performance of energy conservation of absorbed photons up to Q B reduction. Thus, it appeared that in these gametophytes mostly the electron transport chain around PSII was affected. PI ABS was the only parameter of JIP test that differed from the control; therefore, it can be assumed that the effects of Zn200 on the photosynthetic apparatus were rather mild. M. polymorpha demonstrated a substantial resistance to Zn, but also higher plants may effectively respond to this metal: for instance, in Beta vulgaris treated with 50, 100, and 300 µM Zn for 10 days, only the highest concentration showed a marked inhibitory effect on photosynthesis [22]. Nevertheless, M. polymorpha did not seem to suffer damage to the OEC, unlike what occurred with B. vulgaris.

Chlorophyll a Fluorescence Transient Kinetic and OJIP Parameters
The overall functional efficiency of plants was investigated by the analysis of the biophysics of the photosynthetic light reactions. Such evaluation was performed by the measurement of PSII fluorescence. This was recorded, at the aforementioned times, by a chlorophyll fluorometer (Handy PEA, Hansatech Instruments Ltd., Pentney, King's Lynn, UK). Gametophytes were harvested, dried on blotting paper and part (two or three spots) of their surface was darkened with specific clips for 30 min. Nine measurements were then taken for each treatment: two from each of three gametophytes (replications), and three from the fourth replication. The darkened spots were exposed for 1 s to 3500 µmol photons m −2 s −1 (650 nm peak wavelength) and ChlF was recorded. Data were processed by PEA plus software (Hansatech Instruments Ltd., King's Lynn, UK), which performed the analysis of the fast fluorescence kinetics, i.e., JIP test [55]. The recordings from each gametophyte were averaged to yield a single value, which was then treated as an independent replication. The JIP test parameters were calculated from ChlF values recorded at 50 µs, 100 µs, and 300 µs, along with F O , F J , F I , and F M [32]. The parameters are listed in Table 1. and F I ), respectively, and P = peak (maximum fluorescence, F P or F M ). PSI = photosystem I; PSII = photosystem II; RC = total number of reaction centers within the gametophyte spot measured; CS = excited PSII cross section; Q A = primary quinone acceptor of PSII; Q B = secondary quinone acceptor of PSII; OEC = oxygen evolving complex of PSII [56].

Statistical Analyses
The data were first checked for normality of distribution (by Shapiro-Wilk test) and homogeneity of variances (by Levene test). The values of ChlF and the parameters of JIP test were compared between each treatment and the respective control, at each time, by Student's t test. The level of significance was p < 0.05 (*). Statistical analyses were performed by Past 4.06b [57] and graphs were drawn by Microsoft Excel 2016 and GraphPad Prism 9.

Conclusions
Our data demonstrate that high concentrations of metal micronutrients such as Cu, Fe, and Zn impact the photosynthetic machinery of the liverwort M. polymorpha. The mechanisms of action and the extent of negative effects depend on the element and its concentration. Copper, especially at the highest concentration, disrupted the entire electron transport chain, whereas Fe had negative effects mainly around PSII, that were less severe than those of Cu. The effects of Zn were even weaker and of shorter duration.
Given the scarcity of data available on the response of photosynthesis to metal micronutrients in experimental systems other than higher plants, and given the efficiency with which organisms such as M. polymorpha absorb chemical elements from the environment, the present study may represent a starting point for further investigations on the effects of Cu, Fe, and Zn and on the mechanisms underlying heavy metal detoxification and tolerance in bryophytes. This represents basic knowledge to elucidate the evolution of the biochemical and molecular processes that confer to land plants resistance to heavy metals and to develop novel and effective biomonitoring techniques for the protection of the environment.